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Modulation of inflammation and oxidative stress in canine chondrocytes

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  • 1 Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762.
  • | 2 Department of Surgery, Charleston Veterinary Referral Center, 3484 Shelby Ray Ct, Charleston, SC 29414.
  • | 3 Department of Biomedical and Chemical Engineering, LC Smith College of Engineering and Computer Science, Syracuse University, Syracuse, NY 13244.
  • | 4 Nutramax Laboratories Inc, 2208 Lakeside Blvd, Edgewood, MD 21040.
  • | 5 Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762.
  • | 6 Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762.
  • | 7 Nutramax Laboratories Inc, 2208 Lakeside Blvd, Edgewood, MD 21040.
  • | 8 Department of Orthopaedic Surgery, School of Medicine, Johns Hopkins University, Baltimore, MD 21239.

Abstract

Objective—To determine whether oxidative stress could be induced in canine chondrocytes in vitro.

Sample—Chondrocytes obtained from healthy adult mixed-breed dogs.

Procedures—Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300μM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1β (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration.

Results—Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide–stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1β and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production.

Conclusions and Clinical Relevance—Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.

Contributor Notes

Presented in part as an abstract at the Annual Meeting of the Veterinary Orthopedic Society, Canyons, Utah, March 2013.

This manuscript represents a portion of a thesis submitted by Dr. Dycus to the Mississippi State Department of Clinical Sciences as partial fulfillment of the requirements for a Master of Science degree.

Supported in part by Nutramax Laboratories Inc.

The authors thank Dr. Reinhard Grzanna for help with immunocytochemical experiments and analyses.

Address correspondence to Dr. Dycus (dldycus@gmail.com).